Abstract
Elevated serum phosphorus is a major risk factor for vascular calcification, which is characterized by the presence of calcium phosphate deposits, mainly hydroxyapatite crystals. In vitro studies of phosphate-induced calcification show that vascular smooth muscle cells undergo calcification with features similar to those observed in pathological vascular calcification in vivo, including the presence of hydroxyapatite crystals. Here, we describe the double-collagenase digestion method for isolating vascular smooth muscle cells from aorta, and a method for inducing calcification in vitro using high phosphate concentration.
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References
London GM, Drueke TB (1997) Atherosclerosis and arteriosclerosis in chronic renal failure. Kidney Int 51(6):1678–1695
Rutsch F, Nitschke Y, Terkeltaub R (2011) Genetics in arterial calcification: pieces of a puzzle and cogs in a wheel. Circ Res 109(5):578–592
Monckeberg JG (1902) Uber die reine mediaverkalkung der extremitatenarterien und ihr verhalten zur arteriosklerose. Virchows Arch Pathol Anat 171:141–167
Villa-Bellosta R, Rivera-Torres J, Osorio FG et al (2013) Defective extracellular pyrophosphate metabolism promotes vascular calcification in a mouse model of Hutchinson-Gilford progeria syndrome that is ameliorated on pyrophosphate treatment. Circulation 127(24):2442–2451
Shanahan CM, Cary NR, Metcalfe JC et al (1994) High expression of genes for calcification-regulating proteins in human atherosclerotic plaques. J Clin Invest 93(6):2393–2402
Tintut Y, Patel J, Territo M et al (2002) Monocyte/macrophage regulation of vascular calcification in vitro. Circulation 105(5):650–655
Villa-Bellosta R, Millan A, Sorribas V (2011) Role of calcium-phosphate deposition in vascular smooth muscle cell calcification. Am J Physiol Cell Physiol 300(1):C210–C220
Alfrey AC, Ibels LS (1978) Role of phosphate and pyrophosphate in soft tissue calcification. Adv Exp Med Biol 103:187–193
Block GA, Hulbert-Shearon TE, Levin NW et al (1998) Association of serum phosphorus and calcium × phosphate product with mortality risk in chronic hemodialysis patients: a national study. Am J Kidney Dis 31(4):607–617
Giachelli C, Bae N, Lombardi D et al (1991) Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR). Biochem Biophys Res Commun 177(2):867–873
Boström K, Watson KE, Horn S et al (1993) Bone morphogenetic protein expression in human atherosclerotic lesions. J Clin Invest 91(4):1800–1809
Jono S, McKee MD, Murry CE et al (2000) Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 87(7):E10–E17
Villa-Bellosta R, Sorribas V (2009) Phosphonoformic acid prevents vascular smooth muscle cell calcification by inhibiting calcium-phosphate deposition. Arterioscler Thromb Vasc Biol 29(5):761–766
Steitz SA, Speer MY, Curinga G et al (2001) Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res 89(12):1147–1154
Proudfoot D, Skepper JN, Hegyi L et al (2000) Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res 87(11):1055–1062
Ewence AE1, Bootman M, Roderick HL, Skepper JN, McCarthy G, Epple M, Neumann M, Shanahan CM, Proudfoot D (2008) Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization. Circ Res. 2008 Aug 29;103(5):e28-34. doi: 10.1161/CIRCRESAHA.108.181305. Epub 2008 Jul 31.
Shroff RC, McNair R, Figg N et al (2008) Dialysis accelerates medial vascular calcification in part by triggering smooth muscle cell apoptosis. Circulation 118(17):1748–1757
Acknowledgements
R.V.-B. is supported by a Juan de la Cierva postdoctoral contract from the Spanish Ministry for Economy and Competitiveness (JCI-2011-09663) and M.R.H. by an FPI predoctoral Fellowship from the Spanish Ministry for Science and Innovation (BES-2011-043938).
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Villa-Bellosta, R., Hamczyk, M.R. (2015). Isolation and Culture of Aortic Smooth Muscle Cells and In Vitro Calcification Assay. In: Andrés, V., Dorado, B. (eds) Methods in Mouse Atherosclerosis. Methods in Molecular Biology, vol 1339. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2929-0_8
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DOI: https://doi.org/10.1007/978-1-4939-2929-0_8
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2928-3
Online ISBN: 978-1-4939-2929-0
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